A Montage of Genetic Modification

1953: Watson, Crick and Franklin discover the structure of DNA.

1973: Boyer and Cohen create the world’s first ever GMO when they modify the bacteria E. coli to express an antibiotic-resistance gene. In the process they unintentionally foreshadow a serious problem soon to hit the world: the evolution of antibiotic-resistant bacteria in hospitals.

1974: Jaenisch and Mintz create the first GM animal. They injected a primate virus into mouse embryos, then transplanted the embryos into surrogate mothers. The mice grew up normally except that they contained the viral DNA.

1978: Genentech, the world’s first genetic engineering company is founded, and engineers E. coli that can produce human insulin. Diabetics and livestock everywhere rejoice.

1980: The U.S. Supreme Court rules 5 to 4 in General Electric’s favour that “A live, human-made micro-organism is patentable subject matter”. In so doing, it sets the entire course of GMO history to come. GE immediately patents a bacteria engineered to eat crude oil.

1983: The first modified plant is created, again by adding an antibiotic resistance gene. Can you guess the species? (Hint: it was the ’80s). Yep, of course it was tobacco.

1994: Calgene produces the first commercial genetically modified (GM) crop plant, the Flavr Savr tomato. This tomato doesn’t produce a natural protein that degrades cell walls, meaning it stays ripe for longer. The Flavr Savr experiences a tumultuous commercial life of initial success, then by a decline at the hands of consumer distrust, and finally discontinuation by 1997.

1995: The commercial GMO market explodes, with the development of potato, cotton and maize strains that can resist insects.

In the ensuing two decades, two particular classes of modification have come to dominate the GM plant market: insect resistance (via insertion of the “Bt” toxin gene), and resistance to the herbicide “glyphosate” (marketed as Roundup). Glyphosate resistance now dominates the GM market to such a degree that it is present in a whopping ~90% of all transgenic crops, making it the Big Cheese of commercial GMOs. We’ll talk about this as well as Bt in the next instalment.

How many GMOs are out there?

To date, all GMOs approved for human consumption have been plants. A common source of confusion regarding this claim is recombinant bovine growth hormone (rBGH), which is injected into dairy cattle to increase milk production. rBGH is produced by genetically modified bacteria, in much the same way as human insulin. Injecting rBGH into cattle doesn’t cause them to become genetically modified. It is however a form of doping, one which is demonstrably harmful for their health and wellbeing. Human growth hormone has been abused by athletes since the ’80s.

Nonetheless, there’s also a clear legislative bias at play against commercialising GM animals. This may reflect an unproven notion that there’s less risk of GM plants escaping and spreading. A more reasonable argument might be that because plants lack sentience, there’s no risk of them suffering because of a modification. The main reason for the bias may not be so rational though.

Since animals are our closest evolutionary ancestors, we typically hold them in a more reverential and even “sacred” light than plants. You can probably imagine a mutant two-trunked pine tree without being too bothered, but a two-headed rat feels a lot more uncanny valley.

Whatever the reason, at this point in history, crop plants are the undisputed stars of GM technology, so we’ll refocus our radar in the direction of agriculture.

Delicious Data About Agriculture

Agriculture covers a full third of the Earth’s land area, and as of 2013, GM crops made up about 3.5% of the total. That corresponds to more than 1.7 million square kilometers, or an area greater than the entire landmass of Iran. Given this, it’s probably fair to say the prevalence of GMOs is not insignificant.

Legal regulations and social attitudes towards GMOs vary widely between countries, which means that these crops aren’t just scattered around the globe randomly. A particularly rich source of information on GM crops is the report Global Status of Commercialized Biotech/GM Crops: 2012, commissioned by the pro-GM group ISAAA (The International Service for the Acquisition of Agri-biotech Applications). Despite their partisanship on the issue the data seem solid, and the report is worth a read if you’re interested in details about a particular country’s GMO activities.

Which nations are the biggest adopters of GMOs? There were only 28 countries growing GM crops as of 2012, though these countries are home to 60% of the world’s population. Uptake is overwhelmingly focused in North and South America. Interestingly, and largely owing to Europe having the strictest GMO regulations in the world, there are only eight industrialised countries growing GM crops, meaning the rest are developing nations.

Most GM-growing nations are currently focusing on cotton, maize and soybean. GM food crops are predominately used as livestock feed rather than for human consumption, and as mentioned earlier, most GM crops are herbicide resistant and/or insect resistant. This is changing though, with an increasing proportion of “second generation” strains entering the market, which have these traits stacked with others, such as enhanced nutrition or drought tolerance. The USA and China are cultivating GM versions of several other food crops, including things like papaya, sugarbeet and sweet pepper.

While the USA has the greatest land area devoted to GM crops of any nation, as well as the highest number of GM species, GM land is mostly devoted to just a few staple crops, for which an extremely high proportion grown are GM varieties. For the “big three” of cotton, maize and soybean, over 90% of farms are now growing GM varieties. In Canada, a record high of 97.5% of canola crops are GM.

The increasing uptake of GM crops is an interesting story. Despite the USA easily dominating the pack in this modern day space race, the vast majority of remaining GM crops – and 90% of GM farmers – are located in the developing world. Developing nations are also taking up GM technology at a greater rate. As you can see in the chart below, industrialised nations have already lost the majority share of the market.

How do we explain the huge differences in GMO legislation and uptake rates between countries, particularly Europe and the USA? It’s worth first reminding ourselves that, by many metrics, the USA is just a weird outlier, so this may be a very difficult question to answer.

Nonetheless, one possible explanation is labelling requirements (though the causality is hard to tease apart). In the late ’90s, a strong opposition movement to GMOs grew in Europe, and it succeeded in mandating strict labelling of any GM products. Supermarkets responded with a wave of panic, banning products containing any GM ingredients out of fear of losing customers. In a very short time, the entire European GM industry was dead. Conversely, see North America on the graph below (click for larger version). It has no labelling requirements.

Without labelling of GM products, there is less consumer concern and less avoidance of them, meaning the economic incentive for farmers is to grow GM crops rather than less efficient conventional ones. Is this a bad situation for the USA? This debate is currently raging in several US states, with recent or upcoming votes on GMO labelling. All we will say here is that when public concern is coupled with scientific misunderstanding, the outcome can be quite harmful.

Unravelling Some Sticky Side-Issues

Neil deGrasse Tyson was recently lambasted for defending GM technology by claiming it is not all that different from the domestic selection that humans have been exerting on plants and animals for thousands of years. As he pointed out when he later clarified his statement, there is a big sticky mess of related issues tangled up with GMOs, and it was these that his attackers mostly took issue, not the science itself. It’s worth dissecting out a couple of these confounding topics before closing the book on current GMO status.

The sticky mess includes things like: corporate exploitation of small farmers, monocultures, and the merits of “organic” farming (a term that every organic chemist will tell you is meaningless as they sigh into their erlenmeyer flask).

1. Corporate exploitation and patenting. Tales are rampant of farmers in developing countries being forced into unfair annual contracts for GM seeds, or of organic farmers losing their organic licence then being sued because their crops have been contaminated by a GM strain. Such situations rightly invoke our moral outrage. However, according to the excellently researched and independent Genetic Literacy Project, these stories simply aren’t true. Some are myths while others have the facts twisted. Even if these tales were true though, lawsuits and rigid contracts are issues of equitable IP legislation, not of science. The same problem applies to the pharmaceutical industry, with potentially life-saving medications being fiercely protected by patents and kept artificially expensive.

2. Monocultures. A common claim is that GM crops are always “monocultures”, meaning genetically identical plants are grown en masse. The risk here is that if a virus or pest evolves which one plant is susceptible to, all would be susceptible, leading to rapid losses of huge numbers of plants. As it turns out though, when a GM plant is developed, the trait is typically bred across into many cultivars in order to increase the genetic diversity and minimise this risk. That said, growing only one type of crop in an area does harm soil quality and biodiversity and so should be avoided where possible. Most GM crops, excepting pesticide resistant ones, can be grown in mixed plots with no barriers.

3. Organic food. The main point to stress here is that GM crops are not the opposite of “organic” crops. While organic farming excludes the use of GMOs on ideological grounds, it is primarily an alternative to conventional large-scale agriculture. You could grow a patch of GM alfalfa using entirely organic farming practices if you wanted to. Despite this, GMOs and organics are often pitted against each other in the context of food production and security.

Whatever merits organic farming may have, superior food production is sadly not one of them. A 2012 meta-analysis published in that most weighty of scientific journals, Nature, found that organic farming typically produces 34% lower yields when compared to conventionally farmed crops in comparable conditions. This entertaining and well-researched video explores the pros and cons around organic food and dispels some common myths.

If you’ve made it this far, congratulations! You should now be clued up on exactly what genetic modification means, where GMOs come from, their history, and what the heck is out there at the moment. This means it’s time to face the upcoming last part in the series: GMOs Pt 4: Is the Apocalypse Nigh?

Disclaimer: Trading Atoms has no interests, financial or otherwise, in any biotechnology or related company.

In Part 1 of this series we delved into the realm of genetics and looked at just what constitutes a genetically modified organism (GMO). We said the essential difference is that a GMO usually produces one or more extra proteins that don’t exist in the original species. These extra proteins were added to create some kind of desired trait, such as pesticide resistance in wheat. In the coming instalments we’ll look at the prevalence of GMOs and whether we should be terrified, but before all that dry reality, it’s time to make our very own!

The fabled Umbuku lizard.

Step 1: Pick your species and desired trait

Back in the idealistic days of my childhood, I had a vision for what my life’s work would be: I would be the one to engineer the world’s very first actual Pokemon! It would probably look something like this.

However, as the years rolled by I gradually came to accept the harsh truth: I would never achieve my dream. The problem was that Pokemon tended to violate the laws of physics. And that was before even considering the technical limitations to genetic engineering. So with this lesson of genetic hubris in mind, what kinds of creatures could we build?

Until quite recently, the limited tools at our disposal for manipulating DNA meant that the best we could aim for was the addition or subtraction of maybe a few genes.

But if the sky’s the limit when it comes to DNA manipulation, why haven’t we seen this kind of stuff? Let’s assume that scientists are bound by absolutely no ethical qualms or regulatory oversights, and would be keenly interested in adding a digestive tract and muscular system to the common carrot.

The reason, it turns out, is that the way embryos develop is really, really complicated. To make something as complex as a limb, thousands of different genes have to be turned on and off in precisely the right moments in the right cellular locations and at the right levels. Embryonic development is a splendidly complex genetic symphony. Just look how confusing and boring the development of a fruit fly is!

And that’s a highly simplified explanation, only looking at the very first cell. As you can imagine, the process gets exponentially more complex as different types of cells and tissues begin developing and talking to one another. It quickly reaches the point where a detailed understanding is nearly impossible. The complete story though, if we do ever one day manage to unravel it, looks to be quite beautiful:

So, now that our wildest dreams have been crushed for the foreseeable future, what are we left with?

Well we can still do a lot of pretty interesting things, provided it only involves fiddling with simple and well-understood systems. Generally speaking, this means we’re still limited to changing one or a few genes at a time. While no one is going to be adding wings to lions any time soon, some noteworthy innovations have still been made.

One of the earliest breakthroughs, taking place as early as 1978 and providing a major boon for type 1 diabetics, was the insertion of the human insulin gene into E. coli. Before this time, insulin could only be harvested from the pancreatic glands of slaughtered pigs and cattle – not a cheap or pleasant process for anybody. These days, E. coli bacteria happily grow away in vats churning out the stuff.

With climate change increasingly impacting upon the yield and yearly predictability of agricultural harvests, drought-resistant wheat may soon prove an important tool in the fight for food security, not to mention farmers’ livelihoods. The wheat is being developed right now.

The first genetically modified animal proposed for human consumption is the AquAdvantage salmon. It possesses an extra growth hormone gene that came from a related species of salmon. This extra growth hormone causes it to reach full size in about half the time of a conventional salmon.

While all these developments are clearly useful and quite interesting, none of them are very visually exciting. So, without any further delay, let’s see if we can make a cat that glows in the dark. If all goes to plan, here’s what our GlowKitty might look like:

Step 2: Figure out how to obtain your trait

Fiddling with an entire biochemical pathway is Hard, but luckily for us, the modification needed to make a GlowKitty is actually quite simple – we only have to add a single gene. This gene will make a protein called Green Fluorescent Protein (GFP), which looks a bit like a microscopic barrel. The barrel works by absorbing high-energy blue light and re-releasing it as green light. As long as the gene is turned on in enough of the kitty’s cells, we should get a good healthy glow. Note: the gene that makes GFP is also named GFP. This can be a little confusing, but it’s standard practice in the world of genetics.

But where do we get this handy gene from? GFP originally comes from a handsome bioluminescent jellyfish which lives off the coast of North-Western America. Its name is Aequorea victoria, the Crystal Jelly.

As an aside, GFP has probably been played with more than any other gene in history. If you hadn’t heard of it before, you can find it cited in thousands upon thousands of papers. The extremely handy thing about GFP is that you can stick it onto another protein that you’re interested in. Usually, trying to look at a protein in a cell is like trying to spot a black plane in the night sky. Adding GFP is like installing a navigation light.

So, back to the project at hand. We’ve picked our species (cat), decided what trait we want to give it (glow in the dark), and we know we can get the trait by adding a single jellyfish gene (GFP). Time to move on to…

There’s also a smaller-scale, less sexy type of cloning you can do: simply copying a piece of DNA. It still counts as cloning! You’re replicating a biological sample aren’t you? It turns out that this kind of cloning is way easier than creating a whole living creature. In fact, it’s an extremely common and straightforward lab procedure, and cloning GFP will be our next step in making GlowKitty.

The process used is called Polymerase Chain Reaction (PCR). If you’re not familiar with PCR, it’s a bit too detailed to explain properly here. Basically though, it involves mixing DNA with enzymes and repeatedly heating and cooling the mixture to help the enzymes copy the DNA. This video provides a pretty good insight into what goes down in the lab whenever somebody does PCR:

PCR is an amazingly versatile technique. As the song scientific video explains, it’s central to a whole bunch of DNA-related techniques, from paternity testing to detecting mutations and forensic investigations.

Now we’ve covered the theory, you should get out your PCR machine, turn it on and have it idling at about 90-100ºC. If you don’t have a PCR machine, you can substitute in an oven, a bowl of ice and a pair of tongs. Then just follow these easy steps:

Prepare some DNA containing the jellyfish GFP gene.

Add a dash of DNA-copying enzymes (known as “polymerases”). These can be harvested from bacteria, or really any living creature. Make sure to use only trusted species, as cheaper options can result in mutations. Pyrococcus furiosus makes a product that you can count on for peace of mind.

Season with loose DNA bases, salts and primers.

Cook for about two hours, cycling between hot and cool.

Et voila! If all has gone to plan, you should now have several billion copies of your GFP gene.

Step 4: Put the gene into your species

We hit an immediately problem here: we can’t just inject the GFP gene into an adult cat. If we did so it would only end up in a few cells, and we want our cat to be glowing all over. We’d also like it to one day be able to have GlowKitties of its own, so we need the gene to be in its sex cells too.

The only option we have is to get the GFP gene into a single-celled embryo. This way, the GFP will join the rest of the cat’s DNA, and when the embryo grows and divides, the GFP gene will get copied into every cell too. So, go out and get your hands on some cat embryos.

There are a range of approaches we could try in order to get our gene in there. Injecting it into embryos with a tiny needle is pretty tedious and finicky, but it does seem to work quite well for a lot of species. We could also try chemicals. There are compounds that punch holes in the outer “skin” of cells, allowing our gene to slip in. The problem is that, unsurprisingly, this tends to seriously weaken the embryos. There are other types of chemicals that wrap DNA up in a ball of fat, allowing it to slip right through the embryo’s skin like a ghost through walls. Unfortunately, these chemicals also tend to be a bit toxic.

Yes, that is literally a gene gun. Or if you like, “biolistic particle delivery system”. It fires tiny balls of some kind of heavy metal, often tungsten or gold, which are coated with DNA, right into cells. It works pretty well for plants and animal tissues, where there are a bunch cells together to take the impact. However, as you can imagine, blasting a defenceless little cat embryo with balls of tungsten is like cannonballing a ship. Not good.

As with most things, evolution itself has devised a more elegant solution than anything us humans have been able to come up with. Viruses and certain bacteria have spent billions of years mastering the art of slipping inside living cells. Luckily for us, it’s not too hard to harness these clever critters to do our bidding. We simply have to take out their genetic material and replace it with the GFP gene, and we’ve made the perfect little Trojan horse.

Whichever technique you end up choosing, hopefully you’re successful and get the gene in there.

Step 5: The agonising screening process

After all this rigmarole, we might still only be halfway to having our GlowKitty! It’s time to carry out a bunch of screens and checks, not to mention then raising our embryo to an adult cat.

Most life forms have state-of-the-art defence systems to stop new genes from sneaking into their DNA – after all, that’s the kind of nefarious thing that a virus might try to pull. These defences can also make it quite hard for us to get DNA to stay in an embryo. Depending on what technique was used, we might have to screen hundreds or even thousands of embryos to find one that has taken up the gene. This process can be pretty exhausting, especially in something as complex as a cat. Sooner or later though, we should have our glorious eureka moment.

The GFP gene will have picked a spot somewhere along the cat’s DNA to bury in and join the family. Again depending on what technique was used, the spot was probably picked completely randomly. If we’re lucky, it will have picked a boring patch of DNA that wasn’t doing anything. If we’re unlucky though, it might have dived right into one of the cat’s genes and messed it up. There’s also the chance that two or three copies of GFP have jumped in, all at different spots. We definitely need to investigate this, and we do so by reading the DNA code on either side of the GFP gene.

We can compare these DNA sequences to the cat genome to see where the GFP has buried in. If there are no cat genes in these areas, we can be happy that the GFP hasn’t screwed up anything and push ahead. Otherwise, it’s back to the screening process to find a different glowing embryo.

As the cat develops, we’ll have to monitor that the gene is making enough GFP – but not too much – that it’s making it in the right tissues, and that nothing else unexpected has gone wrong. With a bit of luck though, the cat will grow to term happy and healthy and glowing green.

If you’ve made it up to here: Congratulations. You have obtained your GlowKitty.

scienceinseconds.com

The Twist

The story is that U.S. researchers wanted to study the cat version of HIV (called “Feline Immunodeficiency Virus”, FIV). They did this by adding a resistance gene again FIV, and joined it to GFP to act as a beacon. They followed the same process that we have, choosing a virus to get the genes into cat embryos. These cats can now glow in the dark, and won’t get AIDS as easily.

If you’re the type to be upset by this kind of manipulation of animals, I’ve got some bad news for you: GlowKitty is by no means a unique development. For what it’s worth though, glowing in the dark is not thought to cause any pain or emotional distress, and GlowKitties can lead essentially normal lives, probably oblivious to their sciencey superpowers.

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Stay tuned for GMOs Pt 3: What the Heck is Out There? We’ll be investigating the prevalence and types of modified creatures that have most come to populate the planet.